Process for preparing a polymer/biological entities alloy

ABSTRACT

The present invention relates to a process for preparing a polymer/biological entities alloy, comprising a step of mixing a polymer and biological entities that degrade it, during a heat treatment, said heat treatment being performed at a temperature T above room temperature and said biological entities being resistant to said temperature T, characterized in that said biological entities are chosen from enzymes that degrade said polymer and microorganisms that degrade said polymer.

BACKGROUND OF THE INVENTION

The invention relates to a process for preparing polymer materialsderived from the petrochemical industry and/or biosourced, comprising intheir composition biological entities chosen from enzymes andmicroorganisms enabling them to be degraded.

Polymer materials have been the subject of intensive use in recentyears, in particular in the field of plastics. This intensiveexploitation for common uses has been reflected by an accumulation ofplastics in our environment, which is a source of visual nuisance,congestion of refuse sites and pollution of soils and marine media.Thus, as a result of their intrinsic properties, especially theirresistance to degradation, the treatment of waste derived from thesematerials currently constitutes a real environmental and economicproblem.

Several solutions have been proposed, among which are biodegradableplastic materials. Their formulation is in particular directed towardbeing adapted to degradation by microorganisms of the environment.However, this degradation generally takes place partially. In addition,it requires extremely favorable conditions detailed especially instandard EN 13432. These conditions are encountered under artificialconditions, such as industrial composts. For example, these materialsare generally degraded at temperatures above 40° C. These temperatureconditions are expensive to put in place, from an energy and also afinancial point of view.

The standard alternatives for processing waste, such as incineration ordumping in a refuse site, prove to be detrimental even when they areapplied to biodegradable polymer materials, since their degradation doesnot take place totally.

In addition, biodegradable materials have impaired physical properties,especially in terms of resistance to moisture, to temperature and tomechanical elongation. These deficiencies make them unsuitable for usein standard plastics processing operations such as injection molding orextrusion, and incompatible with the targeted applications.

Thus, these biodegradable materials, although promising, do not satisfythe requirements of industrialists and environmental requirements.

Materials consisting of polymers supplemented with a plant-based fillerto improve the degradability of said materials have also been proposed.However, this degradation was due only to the degradation of saidplant-based filler, which necessarily leads to a partial degradation. Inaddition, such a solution proved to be insufficient since it does notallow the mechanical properties of the polymer to be preserved. Such amaterial is thus also limited as regards its uses in the field ofplastics processing.

There is thus a need for biodegradable materials, which have mechanicalproperties equivalent to those of plastics of petrochemical origin andwhich are suitable for use in the standard operations of plasticsprocessing, said materials being able to be degraded totally, at anacceptable rate of degradation, and of doing so under temperature, pHand humidity conditions that are compatible with those generallyencountered in the natural environment.

SUMMARY OF THE INVENTION

After long, in-depth studies, the inventors have developed a process forpreparing polymer materials comprising in their composition biologicalentities that enable them to be degraded. The polymer materials, orpolymer/biological entities alloys, thus obtained have physicochemicalproperties that enable total degradation under aqueous conditions whileat the same time remaining stable under solid conditions.

The invention thus relates to a process for preparing apolymer/biological entities alloy, comprising a step of mixing a polymerand biological entities that degrade it during a heat treatment, saidheat treatment being performed at a temperature T above room temperatureand said biological entities being resistant to said temperature T,wherein said biological entities are chosen from enzymes andmicroorganisms that degrade said polymer. The inventors have shown,surprisingly and unexpectedly, that such a process allows the inclusionof biological entities into the very structure of the solid polymer,while at the same time maintaining an enzymatic activity or degradingactivity of said biological entities.

Without wishing to be bound by theory, when said biological entities isan enzyme, the inventors put forward the hypothesis that the increase intemperature is accompanied by a vaporization of the hydroxyls of thepolymer, making the enzyme active. Such an activation of the enzymegives rise to a start of hydrolysis of the polymer and thus tovitrification coating said enzyme. The enzyme is then protected.

The inventors have also shown that the presence of the biologicalentities substantially improves the degradability of said alloy, anddoes so without impairing the mechanical properties of the polymer.Thus, the mechanical properties of said alloy are very similar, or evenidentical, to the mechanical properties of the polymer alone. Theseproperties may be determined by means of measuring the resilience, themelt flow index, the tensile parameters such as the maximum tensilestress, the elongation at break or the tensile Young's modulus. Thus,the alloy obtained according to the process of the invention is highlysuitable for the standard operations of plastics processing.

The inventors have shown, surprisingly, that the biological entitiesmaintain its enzymatic activity in the alloy of the invention. Inaddition, said alloy remains stable when it is not in solution. Thebiological entities thus become active only when the alloy of theinvention is placed in solution. Thus, the presence of these biologicalentities makes it possible to control the conditions and the rate ofdegradation of the alloy of the invention.

The alloys of the invention thus have the advantage of being stable whenthey are not placed in solution, which, on the one hand, facilitates thestorage and transportation of the material, and, on the other hand,indicates the existence of a “release” effect (or delay effect) of theactivation of the biological entities. Such a release effect is veryadvantageous since it makes it possible to control the activation of theenzymatic activity and of the degradation of the alloy of the invention.

The terms “polymer/biological entities alloy” and “alloy of theinvention” mean a polymer comprising in its composition biologicalentities enabling degradation thereof. This expression thus encompassespolymer/enzyme alloys, polymer/microorganism alloys andpolymer/enzyme/microorganism alloys. The term “biological entities”means an enzyme or a microorganism. In the context of the presentinvention, said biological entities has the feature of being able todegrade a polymer of interest.

The term “polymer/enzyme alloy” means a polymer comprising in itscomposition enzymes enabling degradation thereof. Preferentially, saidpolymer is a biodegradable polymer. The use of a biodegradable polymeras starting material for performing the process makes it possible toobtain a polymer/enzyme alloy with more advantageous degradationproperties.

The term “polymer/microorganism alloy” means a polymer comprising in itscomposition microorganisms enabling degradation thereof. Typically,these microorganisms produce enzymes that degrade said polymer.

The term “heat treatment” means all polymer transformation operationsduring a raising of the temperature of said polymer, preferentially to atemperature above room temperature, and more preferentially to atemperature above 50° C. Preferentially, said heat treatment allows theinclusion of the biological entities. More preferentially, said heattreatment consists of an operation chosen from extrusion,injection-molding, thermoforming, rotary molding, compression,calendering, ironing, coating, stratification, expansion, pultrusion,extrusion blow-molding, extrusion-swelling and compression-granulation.These operations may be performed on polymers in liquid and/or solidform. In a preferred embodiment, the polymer is in solid form. Inanother embodiment, said polymer is in syrup form.

The term “resistant to a temperature” in connection with a biologicalentity means that said biological entity exhibits an enzymatic/degradingactivity after a heat treatment at this temperature.

In the context of the invention, the term “maintaining the enzymaticactivity” means maintaining any degrading activity of the biologicalentities, enabling a degradation of the polymer. In particularembodiments, the degrading activity of the biological entities in thealloy may be increased or decreased compared to their degrading activitybefore the heat treatment.

The term “room temperature” generally means a temperature of between 20°C. and 30° C., and more preferentially a temperature of about 25° C.

The term “extrusion” means the preparation of a polymer in a desiredform, from a material in the form of granules or power, using anextruder. This term encompasses profiled extrusion, extrusionblow-molding, extrusion-swelling and extrusion-calendering.

This extrusion step takes place at the melting point of said polymer.The polymer is then in a partially or totally molten state. Thistemperature thus depends on the nature of said polymer and the nature ofthe biological entities of interest. This temperature may be readilydetermined by a person skilled in the art, in the light of his generalknowledge. Under the action of the temperature and pressure, saidbiodegradable polymer in molten or partially molten form mixes with theother starting materials, and especially the biological entities thatdegrade it. Preferentially, said temperature T is between the glasstransition temperature and the melting point of said polymer. Morepreferentially, said temperature T is the melting point of said polymer.Typically, said temperature T depends on the nature of said polymer andthe nature of the biological entities of interest and is greater than50° C., preferentially greater than 60° C., preferentially greater than70° C., preferentially greater than 80° C., preferentially greater than90° C., preferentially greater than 100° C., preferentially greater than120° C., preferentially greater than 140° C., preferentially greaterthan 150° C. Typically, this temperature T does not exceed 300° C.

The main function of the extruder is to enable, via the action of thetemperature and the pressure, the passage of the polymer through a diewhich is at its end. Typically, an extruder is composed of one or moreheating sheaths having different temperature levels, one or twoArchimedean screws for transporting the material along the sheath, ahopper for feeding into different points material to be extruded, whichis in the form of granules or flour, a more or less complex die which isat the end of the sheath and which gives the desired shape and size tothe plastic material emerging continuously. Preferentially, theextrusion step is performed using an extruder of BC21 twin-screw type,sold under the name Clextral.

The use of this extrusion step falls within the normal competence of aperson skilled in the art. It generally takes place according to thefollowing steps:

-   -   introduction of a mixture of polymer and of biological entities        that degrade it;    -   passage of said mixture into the extruder;    -   output of a rod through a die;    -   cooling of said rod, optionally followed by drying;    -   chopping in the form of regular granules as a function of the        desired form; and    -   drying, preferentially in a rotary oven, at a temperature of        between about 40° C. and about 60° C., and even more        preferentially at a temperature of about 50° C.

Typically, the biological entities/polymer weight ratio is between about0.1% and about 10%, preferentially between about 0.5% and about 8%,preferentially between about 1% and about 6%, preferentially betweenabout 1% and about 5%, preferentially between about 1% and about 4%,preferentially between about 1% and about 3%, preferentially betweenabout 1.5% and about 3%, and even more preferentially this ratio isabout 2%. This ratio is adapted by a person skilled in the art as afunction of the nature of the biodegradable polymer, the nature of thebiological specifies used, and the desired results, especially in termsof degradability of the alloy obtained via the process.

It is an object of the invention to provide a process for preparing apolymer/biological entities alloy, comprising a step of mixing a polymerand biological entities, said biological entities being chosen fromenzymes and microorganisms that degrade said polymer, wherein said alloyis prepared during a heat treatment performed at a temperature T aboveroom temperature, at which the polymer is in a partially or totallymolten state, and wherein said biological entities are able to degradesaid polymer in said alloy. Preferentially, the process of the inventionalso comprises the addition of a substance that can optimize thedegradation capacities of said biological entities. Typically, when saidbiological entities are enzymes, these substances may be cofactors forsaid enzymes, such as divalent cations.

In the context of the present invention, the term “polymer” coverspolymers derived from the petrochemical industry, biosourced polymersand bio-based polymers.

In a particular embodiment, the polymers that are relevant in thecontext of the present invention are derived from the petrochemicalindustry. The advantage of these polymers is the control of thepolymerization process and of the base constituents, which make itpossible to ensure easy transformation. Typically, they are polymerscontaining monomer units comprising hydrolyzable bonds, for instanceesters or amides.

A non-limiting list of these monomers consists of caprolactone,tetramethylene succinate, esters, esteramides, propylene, C₁-C₆hydroxyalkanoates and butylene adipate-co-terephthalate.

A non-limiting list of polymers that are relevant for performing theinvention consists of polycaprolactone, polytetramethylene succinate,copolyesters, polyesteramides, polypropylene, vinyl polymers, poly(C₁-C₆hydroxyalkanoates) and poly(butylene adipate-co-terephthalate),cellulose acetate, poly(butylene succinate) and polyamides, and mixturesthereof.

Typically, said polyamides are aliphatic polyamides chosen from:

-   -   polycaprolactam (PA6),    -   polylauroamide manufactured by opening of the lauryl lactam ring        (PA12),    -   polyundecanamide manufactured from amino undecanoic acid (PA11),    -   polytetramethyleneadipamide manufactured from        tetramethylenediamine and adipic acid (PA 4-6),    -   polyhexamethylene adipamide (PA6-6),    -   polyhexamethylenenonanediamide manufactured from        hexamethylenediamine and 1,9-nonanedioic acid (PA 6-9),    -   polyamide 6.6, sebacic acid—1,6-hexanediamine manufactured from        hexamethylenediamine and sebacic acid (PA6-10), and

polyhexamethylenedodecanediamide manufactured from hexamethylenediamineand 1,12-dodecanedioic acid (PA6-1 2).

Preferentially, said polyamide is polyundecanamide manufactured fromamino undecanoic acid (PA11).

In another mode of the invention, the polymers that are relevant in thecontext of the present invention are biosourced polymers.

The term “biosourced polymer” means a polymer derived from renewableresources. These biosourced polymers are occasionally used incombination with additives such as plasticizers. Among these biosourcedpolymers, biodegradable biosourced polymers are differentiated fromnonbiodegradable biosourced polymers such as:

-   -   polyamides, especially polyamide PA11;    -   polyvinyl chloride;    -   polyethylene; and    -   polypropylene.

Preferentially, said polymer is chosen from polycaprolactone (or CAPA),polylactic acid, polyethylene terephthalate, poly(trimethyleneterephthalate), a C₁-C₆ polyhydroxyalkanoate, cellulose acetate,poly(butylene adipate-co-terephthalate), poly(butylene succinate) andpolyamide PA11 and mixtures thereof.

More preferentially, said polymer is polylactic acid or PLA. PLA hasmechanical properties similar to certain petrochemical thermoplasticssuch as polypropylene.

Even more preferentially, said polymer is polycaprolactone or CAPA.

In another embodiment, said polymer is a bio-based polymer. The term“bio-based polymer” means a polymer manufactured using a compound ofnatural origin, preferentially of non-petrochemical origin.

In the context of the present invention, a biological entities should bechosen, preferentially an enzyme, which withstands the operatingtemperature of the extrusion step according to the process of theinvention. A person skilled in the art has general knowledge enablinghim to determine these temperatures and to identify biological entitiesthat withstand these temperatures.

The term “biodegradable polymer” means a material that can be degradedby biological entities. The result of the degradation of such materialsis the formation of water, carbon dioxide and methane, and optionallybyproducts. The byproducts obtained during the degradation of saidpolymers are nontoxic.

Standard NE 13432:2000 states the requirements relating to packagingthat is upgradable by composting and biodegradation. It sets thecharacteristics that a material should have in order to be defined ascompostable. It is based on the following criteria:

-   -   The biodegradability: it is measured by the metabolic conversion        of the material into carbon dioxide. This property is measured        using the standardized method EN 14046. The degree of        decomposition to be achieved is 90% in less than 6 months.    -   The disintegration: this is the fragmentation of the material        and its absence of visual identification in the final compost.        It is measured by the composting method EN 14045. The material        should be disintegrated in the presence of organic waste in less        than three months. After this time, the compost is screened on a        2 mm screen. The residues of the material greater than 2 mm in        size are considered as undisintegrated. This fraction should        represent less than 10% of the initial mass.    -   A low content of heavy metals and the absence of negative        effects on the quality of the compost: a plant growth test is        performed, according to the method OECD test 208, with a sample        of compost. Other physicochemical parameters of the compost        should not be modified (relative to a compost not containing        polymers): salinity, % of nitrogen, phosphorus, magnesium and        potassium.

DETAILED DESCRIPTION

In a first preferred embodiment, said biological entities are enzymes,preferentially enzymes that are resistant to the extrusion temperature.

The term “enzymes that are resistant to the extrusion temperature” meansenzymes whose protein structure and/or enzymatic activity are notaffected by the temperature at which the extrusion step according to theprocess of the invention is performed. According to the invention, thedegrading activity of the enzymes may decrease but said enzymes remainable to depolymerize the polymer in the alloy. These enzymes are thushighly suitable for use at temperatures above room temperature.

Preferentially, said enzymes are chosen from heat-resistant enzymes andheat-stabilized enzymes.

The term “heat-resistant enzymes” means enzymes whose intrinsic natureaffords resistance to high temperatures, in particular to thetemperature at which the extrusion step according to the process of theinvention is performed. More particularly, a heat-resistant enzyme stillexhibits a degrading activity after the extrusion step according to theprocess of the invention, so that said enzyme is able to degrade thepolymer in the alloy.

Preferentially, said heat-resistant enzymes are chosen from lipase PSfrom Pseudomonas cepacia, lipase AK from Pseudomonas fluorescens, lipaseB from Candida antarctica, proteinase K, a C₁-C₆ polyhydroxyalkanoatedepolymerase, and mixtures thereof.

The term “heat-stabilized” or “heat-protected” enzymes means enzymeswhich are not naturally heat-resistant, but which are in a particularform that gives them resistance to the temperature at which theextrusion step according to the process of the invention is performed.Preferentially, these heat-stabilized enzymes are obtained via achemical or physical process.

Preferentially, said heat-stabilized enzymes are chosen from enzymesencapsulated in nanocapsules consisting of the same material as saidpolymer, enzymes encapsulated in cage molecules and enzymes aggregatedtogether.

These heat-stabilized enzymes may be obtained by encapsulation of thenon-heat-resistant enzymes in nanocapsules, preferentially innanocapsules consisting of the same material as said polymer. Theencapsulation techniques are well known to those skilled in the art.Typically, this encapsulation is performed by using nanoemulsions. Thisencapsulation of the enzymes makes it possible to control the activationof the enzymes. This embodiment of the invention is particularlyadvantageous for a use of the alloys of the invention in the wrapping offood products.

These heat-stabilized enzymes may also be obtained by encapsulation ofthe enzymes in cage molecules. Such an encapsulation makes it possibleto protect said enzymes from any temperature-related degradation.

The term “cage molecule” means a molecule that can be inserted into thestructure of said enzymes to stabilize them and to make them resistantto temperatures above room temperature.

These heat-stabilized enzymes may also be obtained by aggregatingnon-heat-resistant enzymes together. A person skilled in the art hassufficient technical knowledge to perform such an aggregation.

In a particular embodiment, said enzymes are in the form of apoenzymes,and are activated in the presence of cofactors.

In a second embodiment, said biological entities are microorganisms.

Typically, these microorganisms are microorganisms that may or may notbe capable of sporulating and producing enzymes that degrade polymers ofinterest. Preferentially, these microorganisms are bacteria, fungi oryeasts. In the context of the invention, the term “microorganism” alsoincludes spores.

In one embodiment, the alloy of the invention is a polymer/microorganismalloy, preferentially a polylactic acid/microorganism alloy.

Preferentially, said microorganisms are chosen from bacteria of thegenus Ochrobactrum, bacteria belonging to the phylum of firmicutes(Firmus cutis), bacteria of the class of bacilli, especially thebacterial strains Bacillus cereus spp. and more particularly Bacilluscereus G9241, and the bacterial strains Bacillus clausii spp.

More preferentially, said microorganism is a bacterial strain of thegenus Ochrobactrum, known as Ochrobactrum sp. 37S and depositedaccording to the Treaty of Budapest on 23 Jul. 2009 in the name of theCentre National de la Recherche Scientifique at the Collection Nationalede Cultures de Microorganismes under the number CNCM I-4212, or avariant of said strain, said variant being capable of degradingpolylactic acid.

Preferentially, said polymer is polylactic acid and said biologicalentity is Ochrobactrum sp. 37S bacteria. Typically, said bacteriadegrades polylactic acid.

The term “variant” means:

-   -   a natural variant of a strain according to the invention, i.e. a        variant obtained spontaneously from a strain according to the        invention after incubation in a selection medium. A natural        variant is thus obtained without any genetic manipulation by the        operator, but only by natural mutation of the strain and        selection of this mutated strain in a suitable medium, or    -   a variant of a strain according to the invention comprising at        least one mutation in its genome, said mutation being induced by        genetic engineering, for example by site-directed mutagenesis or        random mutagenesis. For example, random mutagenesis may be        performed using mutagens such as radiations (UV rays, ionizing        radiations, heat) or chemical compounds (nitrous acid, ethyl        methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanine,        N-ethyl-N-nitrosourea, acridine orange, proflavin, etc.).

The term “mutation” means the addition, deletion or substitution of atleast one nucleotide in the genome of the strain according to theinvention.

In another embodiment, the alloy of the invention is a polymer/sporesalloy, such as a polylactic acid/spores alloy. In a particularembodiment, the spores are spores of Bacillus licheniformis. Thus, theinvention relates to a process for preparing a polymer/biologicalentities alloy, comprising the following steps:

i) selection of a polymer, preferentially a biodegradable polymer;

ii) selection of biological entities which are capable of degrading saidpolymer and which are resistant to a temperature T above roomtemperature; and

iii) mixing of said polymer and of said biological entities during aheat treatment performed at the temperature T,

characterized in that said biological entities are chosen from enzymesthat degrade said polymer and microorganisms that degrade said polymer.

The invention also relates to a process for preparing apolymer/biological entities alloy comprising a step of extruding apolymer with biological entities selected from the group consisting ofenzymes and microorganisms that degrade said polymer, wherein thebiological entities/polymer weight ratio is between about 1% and 5%, thestep of extrusion being performed at a temperature at which the polymeris in a partially or totally molten state.

All the features described previously apply to this process.

The process has the advantage of being compatible with the standardequipment used in the plastics processing industry, which enables itsrapid and direct implementation by professionals, without significantmodification of the production tools conventionally used, especiallythose ordinarily reserved for the production of plastics ofpetrochemical origin.

In addition, the process of the invention does not require the use ofproducts that are potentially hazardous to human health or to theenvironment. Nor does it generate any byproducts that may represent sucha hazard.

The process of the invention makes it possible to obtain a “tailor-made”polymer/biological entities alloy by including biological entities,preferentially enzymes, with a polymer, preferentially a biodegradablepolymer, which conditions the degradation of the alloys thus obtained.

The invention thus makes it possible to obtain a polymer/biologicalentities alloy, preferentially a sole polymer/enzyme alloy for which itis possible to control the rate of degradation.

A non-limiting list of these polymer/enzyme alloys consists ofpolycaprolactone/lipase PS from Pseudomonas cepacia;polycaprolactone/lipase B from Candida antarctica; polylacticacid/proteinase K;

C₁-C₆ polyhydroxyalkanoate/C₁-C₆ polyhydroxyalkanoate depolymerasecouples.

Preferentially, the invention relates to a process for preparing apolymer/enzyme alloy, comprising the extrusion of polycaprolactone witha lipase, preferentially amanolipase PS from Pseudomonas cepacia, at themelting point of polycaprolactone in an enzymes/polymer ratio of 2% byweight. Specifically, the inventors have demonstrated that the alloythus obtained degrades totally after 4 months in aqueous medium, whereaspolycaprolactone not supplemented with lipase does not degrade withinthis time period.

The inventors have also shown and exemplified that such an alloydegrades at extrusion temperatures of 80° C., 100° C., 120° C. and 140°C.

In a preferred embodiment, the invention also relates to apolymer/enzyme alloy, characterized in that said enzymes degrade saidpolymer and that they are heat-stabilized.

These alloys are highly advantageous due, firstly, to their improveddegradability when they are placed in aqueous solution, and, secondly,to their mechanical properties adapted to their uses in industry. Theinclusion of the enzyme into the alloy does not impair the properties ofthe polymer alone. Thus, the mechanical properties of said alloy arevery similar, or even identical, to the mechanical properties of thepolymer alone.

Preferentially, said heat-stabilized enzymes are chosen from enzymesencapsulated in nanocapsules consisting of the same material as saidpolymer, enzymes encapsulated in cage molecules and enzymes aggregatedtogether.

All the technical features mentioned previously concerning said polymerand said heat-stabilized enzymes are applicable to this alloy.

Alternatively, the invention relates to a polymer/biological entitiesalloy characterized in that said polymer is polylactic acid and saidbiological entities are Ochrobactrum sp. 37S bacteria, said bacteriadegrading polylactic acid.

In another aspect, the invention relates to the use of thepolymer/biological entities alloy of the invention in the sectors ofagriculture, horticulture, packaging, catering, the environment,transport, textiles, electronics and pharmacy. Hereinbelow, the terms“alloy of the invention” and “polymer/biological entities alloy obtainedaccording to the process of the invention” are used withoutdiscrimination.

Preferentially, the invention relates to the use of the alloy of theinvention in the packaging sector, more preferentially for packagingfood products, especially fruit and vegetables, and in the bakerysector. Thus, the invention relates to the use of the alloy of theinvention as a food packaging with a long shelf life. Specifically, thealloy of the invention is highly suited to coming into contact with foodproducts since it does not have any harmful effects, it is readilydegradable and it constitutes a barrier to the passage of carbondioxide, oxygen and water, thus preventing the impairment of foods.

The invention also relates to the use of the alloy of the invention inthe agriculture sector, especially for obtaining granules comprisingplant-protection products, mulching films, tunnel films and tying-upmaterials.

The invention also relates to the use of said alloy for obtainingbottles.

Finally, the invention relates to the use of the alloy of the inventionin the medical field, especially for producing resorbable sutures and asa therapeutically active molecule vector. Preferentially, these alloysare used in the form of nanoparticles containing said therapeuticallyactive molecule. A person skilled in the art has the general technicalknowledge enabling him to prepare nanoparticles from the alloy accordingto the invention.

The advantage of using the alloys of the invention in therapy is obviousin several respects. Firstly, the degradation of the nanoparticle makesit possible to deliver the medicament gradually to the target cells. Inaddition, it makes it possible to limit the side effects associated withthe accumulation of the therapeutic molecule in certain tissues, such asthe detoxification organs such as the liver and the kidney. Finally, thebiodegradability of such nanoparticles limits the potentialenvironmental impacts, which are associated with the spreading of thenanoparticles into the external medium via the natural secretions of thepatient.

The process for preparing the alloys according to the invention makes itpossible to obtain alloys whose degradability is suited to the desireduse. Thus, in a therapeutic context, a person skilled in the art will becapable of adapting the process to obtain a alloy that does not entailany accumulation of nanoparticles in certain organs of the treatedpatient, so as to be able to avoid the adverse effects that may beassociated with the presence of said nanoparticles.

A person skilled in the art will also have to adapt the nature of thetherapeutically active molecule encapsulated by the nanoparticle of theinvention as a function of the type of pathology to be treated and ofthe nature of the nanoparticle per se. Specifically, depending on thephysicochemical properties of said nanoparticle (especially its weight,its size, its lipophilicity, its hydrophilicity and its state ofionization), it may or may not be able to cross the transmembranebarrier. Thus, the nature of the nanoparticle has an influence on thedistribution of the therapeutically active molecule in the individual towhom the nanoparticle consisting of the alloy of the invention isadministered.

EXAMPLES Example 1 Preparation of Polymer/Enzyme Alloys According to theInvention

Materials and Methods

1. Inclusion of Enzymes into the Starting Material During the ExtrusionStep

The incorporation of enzymes into the starting biodegradable polymer isperformed during the “extrusion” step.

The extrusion step is performed using an extruder of BC21 twin-screwtype of Clextral brand (motor power 9 kW, maximum screw speed 600 rpm,maximum current 18.9 A). The screws have a diameter d of 25 mm and theseparation between the two screws is 21 mm. The length of the sheath is600 mm, i.e. a ratio L/d of 24.

The extrusion takes place in 5 steps:

-   -   1. introduction of a biodegradable polymer/enzymes mixture,    -   2. passage of said mixture into the extruder,    -   3. output of a rod through a circular die 3 mm in diameter,    -   4. cooling of the rod in a bath of cold water three meters long,        followed by “drying” with pulsed cold air,    -   5. cutting in the form of regular granules by a system with a        rotating knife.

The formulations may vary as a function of the biodegradablepolymer/enzymes ratio. In the experiments presented in the presentpatent application, the results correspond to a ratio of 2% (m/m) ofenzyme in the material.

The granules obtained by extrusion are then dried in a rotary oven at50° C. for 15 hours (rotary mixer equipped with a jacket withcirculation of oil) so as to remove the residual water present, due tothe passage into the tank of water. The monitoring of the moisturecontent during the drying is performed by means of a moisture analyzerequipped with infrared resistances.

2) Preparation of the Specimens—Injection-molding

In order to evaluate the mechanical properties of the polymer/enzymealloy obtained, and also its degradation capacities, molded pieces ofstandard format, referred to as “specimens”, are obtained byinjection-molding.

Injection-molding is a batch process for putting in form granules ofthree-dimensional finished products. The principle consists in heatingthe plastic material in the feed sheath to bring it in the molten stateto the assay and compression zone in order to inject it into the moldvia a piston. The molded piece is cooled in the mold, and then ejected.

The injection press used is of the Arburg brand, Allrounder1000-420C-250 model.

The injection-molded specimens correspond to type 1 of standard ISO3167.

Result

1. Selection of the Enzymes in Liquid Medium

For the implementation of the process, enzymes capable of degrading thebiodegradable polymer should be identified, so as to identify theappropriate enzymes/material “couple” that will be used during theextrusion step.

We developed two simple tests in order to identify the enzymes ofinterest. These tests are performed on samples of pure material inspecimen or granule form, respectively.

The tests presented hereinbelow were performed on specimens or granulesof polycaprolactone or CAPA (Perstorp; Ref. 6506; 50 000 g/mol). Theinventors evaluated two commercial enzymes, known for their capacity todegrade CAPA: Lipase PS from Pseudomonas cepacia and lipase AK fromPseudomonas fluorescens (Amano, Japan).

a) Tests on Specimens

The specimens of raw polymers placed under biodegradation conditions arespecimens derived from the injection-molding of CAPA granules. All thebiodegradation tests proceed in an oven thermally regulated at 30° C.

The tests presented in this study were performed in a nutrient solution,the composition of which is defined according to standard ISO 14852:1999. Alternatively, the degradation may also be evaluated in FontaineCristalline brand water, i.e. a mineral water of constant compositionmaking it possible to have the same medium conditions.

For these tests, the specimens are maintained fully immersed in thesolution, which is optionally supplemented with enzymes at a finalconcentration of 200 mg/L.

The monitoring of the degradation of the materials is performed bycalculating the loss of mass of the sample after a defined time ofpresence in the medium.

This method uses a series of 13 identical specimens, five of which areoven-treated at 103° C. for 48 hours in order to determine the waterloss of the sample. This step makes it possible to perform a correctionfor the calculation of the loss of mass of the samples placed inbiodegradation. The other 8 specimens are immersed in the degradationmedium and placed in an oven thermally regulated at 30° C. They are thenwithdrawn after a defined time in order to monitor the behavior of thematerial over time. After each withdrawal, the specimens are dried inthe oven at 103° C. for 48 hours and weighed.

The loss of mass is calculated according to the following formula:

${\Delta\; m} = {{\frac{\left( {m_{i} - m_{f}} \right)}{m_{i}}*100} - {\Delta\; m\mspace{14mu}{between}\mspace{14mu}{room}\mspace{14mu}{temperature}\mspace{14mu}{and}}}$103^(∘)  C.

-   With m_(i): initial mass of the specimen    -   m_(f): final mass of the specimen

The results show that the presence of lipase from Pseudomonasfluorescens does not make it possible to improve the biodegradability ofpolycaprolactone since the degree of degradation is substantially thesame as when the enzymes are not present.

On the other hand, with regards the lipase from Pseudomonas cepacia, avery marked difference is observed from the very first withdrawalperformed after 7 days of contact with the medium. Specifically, adegree of degradation of 5% is obtained, whereas over the same time, thesample degraded in the absence of enzyme shows a loss of mass of only0.2%.

Nevertheless, a steady stage is rapidly observed and the degree ofbiodegradation no longer changes. It was therefore decided to change thenutrient solution supplemented with enzymes (still at 200 mg/L ofnutrient solution). This change was made 44 days after the start of thedegradation. This made it possible to observe a resumption ofdegradation up to about 9% in 70 days.

b) Tests on Granules

The advantage of this method is that it makes it possible to work onsmall reaction volumes, making the experiments substantially lessexpensive. Furthermore, several enzymes and several concentrations canbe tested simultaneously. Finally, the possibility of working with highconcentrations of enzymes makes it possible to obtain high degrees ofdegradation over short times.

In this experiment, the CAPA granules (approximate volume: 50 mm³;approximate mass: 40 mg) were incubated in 1.9 ml of a buffer (25 mMphosphate buffer, pH 7, sodium azide 2 g/L), optionally supplementedwith lipase PS from Pseudomonas cepacia at a final concentration of 20mg/ml. The incubation is performed at 28° C. At regular time intervals,the granules are withdrawn, rinsed thoroughly with water and thenincubated for 24 hours at 28° C. for drying. The mass is then determinedand compared with the starting mass, i.e. before incubation in thereaction medium.

The results show a degree of degradation of the CAPA of more than 50%with lipase PS from Pseudomonas cepacia, under the stated conditions,confirming the results obtained with the specimens.

This protocol may be used for the determination of the most relevantenzyme for the purpose of its subsequent inclusion into a given polymer.

Furthermore, it may also make it possible to identify certain“additives” which have the property of optimizing the degradationactivity. These additives may then be added with the enzyme to thematerial during the extrusion step, to accelerate the degradationthereof.

2. Evaluation of the Heat Resistance of the Enzymes in Order toDetermine the Optimum Extrusion Temperature

During the extrusion step, the polymer/enzyme mixture is subjected to ahigh temperature corresponding to the melting point of the polymer. Inthe case of CAPA, we set this extrusion temperature at 80° C. Accordingto the supplier's technical sheet, lipase PS from Pseudomonas cepacia isstable in powder form for several hours at 100° C.

However, these data refer to the lipase activity of the enzyme and donot evaluate the heat resistance of its CAPA depolymerase activity.

We therefore performed the experiment on granules described above, buttreating the enzymatic solution (20 mg/ml of enzyme) for 5 minutes at80° C. The time and temperature selected correspond to thermalconditions close to those encountered during the extrusion step.

The results show that this treatment has a low impact on the CAPAdepolymerase activity of the enzyme. Specifically, the inventorsrevealed only a 10% loss of activity at 7 days. It follows that lipasePS from Pseudomonas cepacia is an excellent candidate for preparing theCAPA/enzyme alloy.

3. Incorporation of Lipase PS from Pseudomonas cepacia During thePolycaprolactone Implementation Process

The inventors thus performed an extrusion of polycaprolactoneincorporating said enzyme. To do this, a mixture of polymer in powderform and of enzyme in powder form is placed in a metering device.

In the experiments that are presented herein, the flow rates of thesetwo metering devices were adjusted so as to obtain a percentage ofenzyme in the material of 2% (m/m). A formulation without enzyme wasalso prepared (control) (Table 1).

TABLE 1 Formulations prepared in this study Mass of Mass of enzyme (g)CAPA 6506 P (g) Formulation (mass %) in the metering device in themetering device 98 m % CAPA 6506 + 30 1500 2 m % lipase 100 m % CAPA6506 0 1500

The extrusion was performed at 80° C., so as to have a temperature abovethe melting point of CAPA (60° C.), but not too high, so as to avoid anyrisk of denaturing the enzyme.

The precise parameters of the extrusion, which was performed under thesame conditions for the two formulations, are presented in Table 2.

TABLE 2 Parameters of the extrusions performed in this study ExtrusionSpeed temperatures BC 21, Pressure, Torque Formulation (zone 1 at die)T/min bar (%) 98 m % CAPA 6506 + 18/20/39/65/ 256 71-74 53-54 2 m %lipase 67/72/68/65/71 100 m % CAPA 6506 20/20/40/65/ 256 73-75 55-6065/69/65/65/65

The term “torque” cited in table 2 corresponds to the relative motorintensity and represents the mechanical energy supplied by the extruderto the extruded material.

The granules obtained were then dried in a rotary oven in order toremove the residual water.

The two formulations then underwent an injection-molding step for thepurpose of obtaining normalized specimens. The injection-moldingconditions were identical for the two materials.

Table 3 shows the settings of the injection press that were used in thisstudy.

TABLE 3 Injection-molding parameters on an Arburg 100/420C/250 pressTemperatures ° C. 40/55/60/80/110 (Feed → Nozzle) Imprint Specimen Assayspeed (v401) m/min 8.0 Assay volume (V403) cm³ 14.3 Counterpressure(p401) bar 150 Assay time (t402M) s 3.9 Injection pressure (p301) bar800 Injection flow rate (Q301) cm³/s 5 Switching (V311) cm³ 5Maintenance pressure (p321) bar 650 Maintenance flow rate (Q321) cm³/s 5Maintenance time (t321) s 30 Cooling time (t400) s 25 Cycle time (t902M)s 65.8 Mattress (V321I) s 1.3 Ejector speed (v602) mm/s 10 Ejector force(F601) kN 5 Nozzle position Detached Mold temperature ° C. 18

The degradation efficacies were evaluated in Cristalline mineral water.Briefly, for each formulation, four normalized specimens (whose masseswere measured beforehand) were immersed in a tank of water (Cristallinereference mineral water). The tanks were then placed in a chamberthermally regulated at 32° C. and ventilated.

A specimen was then withdrawn after 1, 2, 3 and 6 months and weighed(after oven treatment at 103° C. for 48 hours). The degradability of thematerial was measured by means of the loss of mass, calculated as statedpreviously).

The results obtained show a total biodegradability of CAPA when theenzyme was incorporated into the material, at and above 102 days ofincubation of the specimens in water, this demonstrating that the enzymewithstood the heat treatments inflicted during the extrusion andinjection-molding steps.

The inventors have thus shown that an enzyme can effectively beincorporated into a polymer from the extrusion phase while at the sametime maintaining the degradation properties of the polymer.

This approach thus constitutes a simple method, which is easy toimplement and which can be readily adapted to other polymer/enzymecouples for controlling the degradation of materials in natural medium.

Example 2 Characterization of the Alloys of the Invention

The inventors compared the mechanical properties of CAPA alone, of CAPAmixed with a plant-based charge and of a alloy consisting of CAPA andlipase PS from Pseudomonas cepacia and obtained according to the processof the invention.

Materials and Methods

1. Production of the Alloys

The CAPA/lipase PS from Pseudomonas cepacia alloy was obtained using thefollowing starting materials:

TABLE 4 Starting materials used for the formulation of the alloys NameSupplier Definition CAPA 6506 Solvay Polycaprolactone powder (CAPA)Wheat Amo Native wheat flour reference “La Doree” predried Lipase AmanoReference “PS SD”

Step of Granulation by Extrusion

The extrusion step was performed in five steps, using a Clextral BC21co-rotating twin-screw extruder

-   -   introduction of the various elements in the envisaged        proportions by means of a weight-metering device and a        volumetric metering device;    -   melting, kneading, degassing, mixing and placing under pressure        of the material in the successive parts of the extruder;    -   output of a rod through a circular die 3 mm in diameter;    -   cooling of the rod in a tank of cold water three meters long,        followed by “drying” with pulsed cold air;    -   cutting in the form of regular granules by a system with a        rotating knife.

Four formulations were thus prepared (the percentages correspond to masspercentages):

-   -   100% CAPA;    -   98% CAPA+2% lipase;    -   80% CAPA+20% wheat;    -   55% CAPA+45% wheat.

The addition of a plant-based charge (wheat flour) is a standardalternative for increasing the degree of degradation of a polymer.Specifically, the maximum degree of degradation corresponds to thedegradation of the plant-based charge, the polymer used as binder not infact being degraded (see hereinbelow).

The granules obtained by extrusion were then dried in a rotary oven at40° C. for 12 hours so as to remove the residual water present, due tothe passage into the tank of water. The dried granules were then able tobe injection-molded and characterized.

Injection-molding Step

The injection-molding step makes it possible to transform the granulesobtained by extrusion into characterization specimens. The principle ofthe injection molding consists in heating the plastic material in asheath to melt it (plasticization phase) and inject it under pressureinto a mold by means of a piston. The material is solidified and partlycooled in the mold, before being ejected. The press used is an Arburgbrand press of Allrounder 1000-420C-250 model.

The injection-molded specimens correspond to type 1 of standard ISO3167.

Certain “97% CAPA+2% lipase” specimens obtained by injection-moldingunderwent a post-treatment before characterization, consisting ofstorage in a sealed zip-lock bag placed in a closed cardboard box for 1month at room temperature, the object of this step being to evaluate theimpact of storage on the stability of the material, both from the pointof view of its mechanical properties and of its capacity to be degraded.

2. Determination of the Mechanical Properties

Tensile Test

The tensile properties are determined according to the recommendationsdescribed in International standard ISO/R 527 (determination of thetensile properties). These tests should be performed under well-definedconditions as regards temperature, humidity and speed of separation ofthe jaws.

The tensile test consists in imposing an elongation onto a specimen ofinitial cross section So and of useful length Lo. The specimen is fittedat its two ends into jaws. One of these jaws, which is mobile, isconnected to a drive system at a linear speed of travel. The measurementof the work is performed using an electronic force sensor (10 kN).

Three items of information are noted from these tests:

-   -   the maximum tensile stress (Cmax TR; MPa),    -   the elongation at break (in %),    -   the tensile Young's modulus (in MPa).

As indicated in the standard, the tests are performed on five specimensof each batch of material and the results presented are the mean ofthese five determinations. The speed of displacement is set at 50 mm/minand the tensile modulus is calculated between 10% and 50% of the maximumstress value. The tensile testing machine used is sold by Zwick.

Charpy Impact

This method makes it possible to study the behavior of definedspecimens, subjected to impact stresses to estimate the fragility orstrength thereof. For these tests, the inventors used the specimens oftype 1 described previously, unnotched.

The equipment used is an impact pendulum with an energy of 15 joules ofthe Zwick brand. The unit is driven by software which records the valuesmeasured and the analyses. During the test, the specimen is placedhorizontally before the supports and hit with the pendulum at itscenter. The Charpy impact resistance corresponds to the energy absorbedby the breaking of a specimen relative to its cross section before thetest (resilience).

3. Rheological Properties: Melt Flow Rate (MFR)

Standard ISO 1133 prescribes a method for determining the melt flow rate(or MFR) of thermoplastics under defined temperature and pressureconditions. This test makes it possible to determine the grade of thepolymer tested and gives information regarding the capacity of thepolymer to flow through a die for a given temperature (which is animportant parameter for injection molding).

The apparatus used is a Zwick brand extrusion plastometer connected to aprecision balance and driven by specific software capable of determiningthe MFR expressed in g/10 minutes.

During a test, the mixture, heated in the vertical cylinder, is extrudedthrough the die using a charge attached to a piston. Five extrudates arecut at regular time intervals, recovered and then weighed. Theparameters to be set are thus the temperature of the cylinder, theweight of the charge and the time between two cuts.

4. Water Degradation Test

For each formulation, four normalized specimens (whose masses, dried at103° C., were measured beforehand) are immersed in a tank of water(mineral water of Cristalline reference, Leclerc). The tanks are placedin a chamber heat-regulated at 32° C. and ventilated. A specimen is thenwithdrawn after 1, 2, 3 and 4 months and weighed (after oven treatmentat 103° C. for 48 hours). The degradability of the material is measuredby the loss of mass calculated in the following manner:Loss (%)=(total loss of mass−loss of water 103° C.)×100Results1. Mechanical and Rheological Properties

The results obtained are presented in the table below:

TABLE 5 Properties of the materials Tensile tests MFR Cmax ElongationYoung's Break at % Formulation Resilience 150° C./ TR at break modulusthe end of Density degradation (mass %) Post-treatment Kj/m² 2, 16 kgMPa (%) (MPa) traction (g/cm³) at 3 months 100% CAPA 23 14 23 533 400 Nobreak 1.11  1 98% CAPA + 2% lipase 24 14 23 530 450 No break 1.14 71 98%CAPA + 2% lipase Storage for ND ND 26 530 500 No break 1.14 77 1 monthin the dark in a sealed bag 80% CAPA + 20% wheat 24 11 19 532 500 Nobreak 1.16 18 55% CAPA + 45% wheat 21  4  9 286 675 Break 1.22 42

This table indicates that the addition of enzyme does not modify themechanical properties of the CAPA tested here, namely the resilience,the MFR or the set of tensile parameters. This is in very markedcontrast with the polymers consisting of 55% CAPA+45% wheat, for whichall of the mechanical properties are degraded relative to CAPA alone.The mixture 80% CAPA+20% wheat itself makes it possible to conserveproperties similar to that of CAPA alone.

Thus, the alloy of the invention is highly suited to improving thedegradation of CAPA, while at the same time conserving its properties.

2. Degradation in Aqueous Media

The results indicate that CAPA alone is not degraded when it is immersedin an aqueous environment. If the addition of 20% of a plant-basedcharge makes it possible to improve the degradation of the alloy, itnevertheless remains that the maximum degree reached is less than 20%.As has already been indicated, this degradation corresponds to thedegradation of the wheat flour, while the material itself remainsunaltered.

An improvement in the biodegradation is thus directly correlated to anincrease in the percentage of the plant-based charge in the alloy, with,as a corollary, impairment of the mechanical properties.

The results also indicate that the addition of enzymes is very much moreadvantageous than the addition of a plant-based charge, since it inducesthe degradation of CAPA (about 70% after 3 months in water), while atthe same time conserving the mechanical properties of the pure material.

The inventors have thus succeeded in showing that the compositionsobtained have better degradability than with a plant-based charge, whileat the same time maintaining the mechanical properties of the polymer.

Finally, storage in the dark and in a sealed bag of the polymer 98%CAPA+2% lipase does not impair either the mechanical properties or itscapacity to be degraded (comparison of the behavior of the polymers 98%CAPA+2% lipase optionally after post-treatment). The alloy of theinvention is thus stable and is not degraded when it is not in solutionand is thus suitable for storage.

In conclusion, the results as a whole indicate that the process forpreparing polymer/enzyme alloys according to the invention is aparticularly attractive industrial solution for the development ofmaterial with a short lifetime, while at the same time reducing thenegative impacts on the environment that are inherent to thedissemination of residues into the natural medium.

Example 3 Impact of the Temperature on the Biodegradation ofPolycaprolactone (CAPA)

In order to demonstrate that the temperature of transformation ofpolycaprolactone has no impact on its biodegradation, polycaprolactonewas extruded at 170° C. and its biodegradation was evaluated.

3.1. Step of Granulation by Extrusion of Polycaprolactone at 170° C.

The extrusion step is performed as defined in Example 1 (see theMaterials and methods section 1), apart from the fact that the extrusiontemperature in this example is 170° C. and the formulation is composedof 100% CAPA (without enzyme). The extrusion parameters are presented inTable 6.

TABLE 6 Parameters of the extrusion performed in this study ExtrusionSpeed Formu- temperatures BC 21, Pressure, lation (Zone 1 to die) T/minbar Torque (%) 100 m % 20-80-7*170 200 42 38 CAPA 65063.2. Injection Molding

The granules of 100% CAPA (cf. 100 m % CAPA 6506) then underwent aninjection-molding step for the purpose of obtaining normalizedspecimens. The injection-molding step is performed as described inExample 1.

Table 7 presents the settings of the injection press that were used inthis study.

TABLE 7 Injection-molding parameters on an Arburg 100/420C/250 pressTemperatures ° C. 40/55/60/80/100 (Feed → Nozzle) Imprint Specimen Assayspeed (v401) m/min 8.0 Assay volume (V403) cm³ 19 Counterpressure (p401)bar 150 Assay time (t402M) s 4.5-7.5 Injection pressure (p301) bar 800Injection flow rate (Q301) cm³/s 5 Switching (V311) cm³ 5 Maintenancepressure (p321) bar 650 Maintenance flow rate (Q321) cm³/s 5 Maintenancetime (t321) s 30 Cooling time (t400) s 20 Cycle time (t902M) s 60Mattress (V321I) s 1 ± 0.5 Ejector speed (v602) mm/s 400 Ejector force(F601) kN 20 Nozzle position Detached Mold temperature ° C. Cold direct,5° C.3.3. Measurement of the Biodegradation

-   -   Degradation in water at 30° C.: monitoring of the loss of mass

The degradation was evaluated in Cristalline mineral water. Fournormalized specimens (whose masses were measured beforehand) wereimmersed in a tank of water. The tanks were then placed in a chamberheat-regulated at 32° C. and ventilated.

A specimen was then withdrawn after 15 days, 1, 2 and 3 months andweighed (after oven treatment at 103° C. for 48 hours). Thedegradability of the material was measured by means of the loss of mass,calculated as specified in Example 1.

As for the formulation 100 m % CAPA 6506 (see Example 2.), the “loss ofmass” of the specimens prepared from the formulation 100 m % CAPA6506-170 does not show any degradation after 2 months. The monitoring ofthe degradation in water at 32° C. makes it possible to demonstrate thatCAPA alone does not degrade over time in water, when it is extruded attemperatures from 80° C. up to 170° C.

Example 4 Inclusion of 2% Lipase PS from Pseudomonas cepacia in 98% CAPAat 100° C.

4.1. Inclusion of the Enzyme

The inventors performed an extrusion of polycaprolactone incorporatingsaid enzyme. To do this, a mixture of 98% by mass of polymer and 2% bymass of enzyme was placed in a metering device. The amounts used in themixture are presented herein (see Table 8).

TABLE 8 Formulation prepared in this study Formu- Mass of enzyme (g)Mass of CAPA 6506 P (g) lation (mass %) in metering device in meteringdevice 98 m % 30 1500 CAPA 6506 + 2 m % lipase4.1.a. Extrusion

The extrusion was performed at 100° C. The extrusion step wasaccomplished as described in Example 1 (see the Materials and methodssection 1).

The extrusion parameters are presented in Table 9.

TABLE 9 Parameters of the extrusion performed in this study ExtrusionSpeed Formu- temperatures BC 21, Pressure, lation (Zone 1 to die) T/minbar Torque (%) 98 m % 20-80-7*100 200 65 53 CAPA 6506 + 2 m % lipase4.2. Injection Molding

The formulation then underwent a step of injection molding for thepurpose of obtaining normalized specimens. The settings of theinjection-molding press that were used in this study are the same asthose used in Example 3 (see Table 7).

4.3. Biodegradation Measurement

The degradation efficacies were evaluated by degradation in water.

-   -   Degradation in water at 30° C.: monitoring of the loss of mass

The degradation was evaluated in Cristalline mineral water as in Example3 (see section 3.3).

A specimen was then withdrawn after 15 days, 1, 2 and 3 months andweighed (after oven treatment at 103° C. for 48 hours). Thedegradability of the material was measured by means of the loss of mass,calculated as specified in Example 1.

The results obtained show a biodegradability with a loss of mass of 54%after 2 months. This demonstrates that the enzyme withstood the heattreatments inflicted during the extrusion and injection-molding steps.

The inventors have thus shown that an enzyme can effectively beincorporated into a polymer from the extrusion phase at 100° C. while atthe same time conserving the degradation properties of the polymer.

Example 5 Inclusion of 2% Lipase PS into 98% CAPA at 120° C.

5.1. Inclusion of the Enzyme

The inventors performed an extrusion of polycaprolactone, incorporatingsaid enzyme. To do this, the polymer was placed in one metering deviceand the enzyme in another, both being in powder form.

In the experiments that are presented here, the flow rates of these twometering devices were set so as to obtain a percentage of enzyme in thematerial of 2% (m/m) (see Table 10).

TABLE 10 Formulation prepared in this study Formu- Mass of enzyme (g)Mass of CAPA 6506 P (g) lation (mass %) in metering device in meteringdevice 98 m % 30 1500 CAPA 6506 + 2 m % lipase5.1.a. Extrusion

The alloy between the polymer and the enzyme was prepared by extrusionat 120° C. The extrusion step was accomplished as described in Example 1(see the Materials and methods section 1).

The extrusion parameters are presented in Table 11.

TABLE 11 Parameters of the extrusion performed in this study ExtrusionSpeed Formu- temperatures BC 21, Pressure, lation (Zone 1 to die) T/minbar Torque (%) 98 m % 20-80-7*120 200 65 55 CAPA 6506 + 2 m % lipase5.2. Injection Molding

The formulation was then subjected to an injection-molding step for thepurpose of obtaining normalized specimens. The injection-moldingconditions were identical to those of Example 3 (see Table 7).

5.3. Biodegradation Measurement

The degradation efficacies were evaluated by means of the degradation inwater.

-   -   Degradation in water at 30° C.: monitoring of the loss of mass

The degradation was evaluated in water under the same conditionsdescribed in Example 3.

The results obtained show a biodegradability with a loss of mass of27.5% after 2 months. This demonstrates that the enzyme withstood theheat treatments inflicted during the extrusion and injection-moldingsteps.

The inventors have thus shown that an enzyme can effectively beincorporated into a polymer from the extrusion phase at 120° C., whileat the same time conserving the degradation properties of the polymer.

Example 6 Inclusion of 2% Lipase PS into 98% CAPA at 140° C.

The inventors performed an extrusion of polycaprolactone, incorporatingsaid enzyme. To do this, the polymer was placed in one metering deviceand the enzyme in another, both being in powder form.

In the experiments that are presented here, the flow rates of these twometering devices were set so as to obtain a percentage of enzyme in thematerial of 2% (m/m) (see Table 12).

TABLE 12 Formulation prepared in this study Formu- Mass of enzyme (g)Mass of CAPA 6506 P (g) lation (mass %) in metering device in meteringdevice 98 m % 30 1500 CAPA 6506 + 2 m % lipase6.1.a. Extrusion

The extrusion was performed at 140° C. The extrusion step wasaccomplished as described in Example 1 (see the Materials and methodssection 1).

The extrusion parameters are presented in Table 13.

TABLE 13 Parameters of the extrusion performed in this study ExtrusionSpeed Formu- temperatures BC 21, Pressure, lation (Zone 1 to die) T/minbar Torque (%) 98 m % 20-80-7*140 200 59 48 CAPA 6506 + 2 m % lipase-1406.2. Injection Molding

The formulation was then subjected to an injection-molding step for thepurpose of obtaining normalized specimens. The settings of theinjection-molding press that were used in this study are the same asthose used in Example 3, section 3.2 (see Table 7).

6.3. Biodegradation Measurement

The degradation efficacies were evaluated by degradation in water.

-   -   Degradation in water at 30° C.: monitoring of the loss of mass

The degradation was evaluated in Cristalline mineral water as in Example3 (see section 3.3).

A specimen was then withdrawn after 15 days, 1, 2 and 3 months andweighed (after oven treatment at 103° C. for 48 hours). Thedegradability of the material was measured by means of the loss of mass,calculated as specified in Example 1 (see the section Result 1.a.).

The results obtained show a biodegradability with a loss of mass of 4.5%after 2 months. This demonstrates that the enzyme withstood the heattreatments inflicted during the extrusion and injection-molding steps.

The inventors have thus shown that an enzyme can effectively beincorporated into a polymer from the extrusion phase at 140° C. while atthe same time conserving the degradation properties of the polymer.

In conclusion, the inventors have illustrated that the process of theinvention makes it possible to obtain a alloy that is degraded atvarious extrusion temperatures, in particular at 80° C., 100° C., 120°C. and 140° C.

Example 7 Methods for Introducing Lipase

In order to demonstrate the importance of the inclusion of lipase byextrusion on the biodegradation of the corresponding material, variousmethods for introducing lipase were performed on a polycaprolactone.

1: Introduction of Lipase by Extrusion (Test 7a)

A mixture of 98 m % polycaprolactone (CAPA 6506) and 2 m % lipase PS wasextruded at 65° C. under the conditions described in Example 1 (see theMaterials and methods section).

The extrusion parameters are presented in Table 14.

TABLE 14 Parameters of the extrusion performed in this study ExtrusionSpeed Formu- temperatures BC 21, Pressure, lation (Zone 1 to die) T/minbar Torque (%) 98 m % 20-20-40-6*65 250 93 63 CAPA 6506 + 2 m % lipase

The formulation then underwent an injection-molding step for the purposeof obtaining normalized specimens. The settings of the injection-moldingpress are the same as those used in Example 3 (see Table 7).

The degradation efficacy of the injection-molded pieces was thenevaluated by means of the degradation in water, according to the methoddescribed in Example 3.

2. Introduction of Lipase by Spraying onto the Surface ofInjection-molded Polycaprolactone Pieces (Test 7b)

A non-extruded polycaprolactone CAPA6506 underwent an injection-moldingstep for the purpose of obtaining normalized specimens. The settings ofthe injection-molding press are the same as those used in Example 3 (seeTable 7).

An aqueous solution of lipase PS was then sprayed onto the surface ofthe injection-molded polycaprolactone pieces to obtain the equivalent ofa mixture of 98 m % polycaprolactone and 2 m % lipase.

The degradation efficacy of the injection-molded pieces was thenevaluated by means of the degradation in water, according to the methoddescribed in Example 3.

3. Introduction of Lipase into Water Used in the Test of Degradation inWater (Test 7c)

A non-extruded polycaprolactone CAPA6506 underwent an injection-moldingstep for the purpose of obtaining normalized specimens. The settings ofthe injection-molding press are the same as those used in Example 3 (seeTable 7).

The degradation efficacy of the injection-molded pieces reference 7a wasthen evaluated by means of the degradation in water seeded with lipasePS, according to the method described in Example 3. The amount of lipaseadded is equivalent to a mixture of 98 m % polycaprolactone and 2 m %lipase PS.

The losses of mass of formulations 7a, 7b and 7c are collated in thetable below:

TABLE 15 Loss of mass in water of samples 7a, 7b and 7c Lipase PS Refer-introduction Loss of mass (mass %) in water after ence method 15 days 1month 2 months 3 months 7a Extrusion 16 34 64 86 7b Surface 17 21 21 21spraying 7c Seeding of the 15 21 31 32 water used in degradation

These results reveal the fact that when the lipase is introduced byspraying or into the degradation solution, the degradation is lesssubstantial than when the lipase is included into the alloy.

In other words, the inventors have shown that the extrusion step makesit possible to obtain an alloy having better degradability than the stepof dispersion/solution of the lipase.

Example 8 Additional Tests

The starting materials used for these additional tests are as follows:

-   -   polycaprolactone CAPA 6506;    -   wood flour Arbocel C320 supplied by Rettenmaier; and    -   La Dorée wheat flour supplied by AMO.

Various alloys of polycaprolactone, lipase PS and/or plant-based charge(wheat flour or wood flour) were prepared by extrusion under theconditions described in Example 1 (see the Materials and methodssection):

TABLE 16 Composition of the various alloys obtained by extrusion in thisstudy Refer- ence Composition  7a 98 m % CAPA + 2 m % lipase  8 55 m %CAPA + 45 m % wood flour  9 80 m % CAPA + 20 m % wood flour 10 100% CAPA11 55 m % CAPA + 45 m % wood flour

The extrusion parameters are presented in Table 17:

TABLE 17 Parameters of the extrusion performed in this study Extrusiontemperatures Speed BC 21, (Zone 1 to die) T/min 20-20-40-6*65 250

The various alloys obtained were then subjected to an injection-moldingstep for the purpose of obtaining normalized specimens. The settings ofthe injection-molding press are the same as those used in Example 3 (seeTable 7).

The degradation efficacies of the injection-molded pieces reference 7a,8, 9, 10 and 11 were then evaluated by means of the degradation inwater, according to the method described in Example 3. These efficaciesare presented in the table below:

TABLE 18 Loss of mass of samples 7a, 8, 9, 10 and 11 Refer- Loss of mass(mass %) in water after ence Composition 15 days 1 month 1 months 3months  7a 98 m % CAPA + 16 34 64 86 2 m % lipase  8 55 m % CAPA + <1 36 9 45 m % wheat flour  9 80 m % CAPA + <1 4 6 7 20 m % wheat flour 10100% CAPA <1 <1 <1 <1 11 55 m % CAPA + <1 <1 <1 <1 45 m % wood flour

The inventors have thus shown that in order to obtain highbiodegradability, the addition of enzymes is markedly more advantageousthan the addition of a plant-based charge.

Example 9 Preparation of Polymer/Spore Alloy

1) Spore Productions

Spore Production Test MJ1

A 1 ml aliquot of Bacillus licheniformis from the culture collection wasincubated in 100 ml of sterile nutrient broth (3 g of meat extract, 5 gof pepton and 2 g of glucose per liter) in a shaker incubator at 45° C.and 180 rpm for 31 hours. The culture was then transferred to a 5 literfermentor filled with 2.9 liter of the same medium and grown at 45° C.under a 180 rpm stirring at a regulated pH of 6.8. After 143 hours ofcultivation, sporulation was provoked by lowering pH to 4.4 while adding30 ml of a 10 g/l manganese sulfate solution. Dry weight in the solutionwas estimated to 14.8 g/l as a result of 3 measurements withoutfiltration. 50% of the bacteria were sporulated after 48 hours at 45° C.Spores were enumerated by the internally modified malachite greenmethod.

Spore Production Test MJ2

A 2 ml aliquot of Bacillus licheniformis from the culture collection wascultivated in 200 ml of sterile nutrient broth (3 g of meat extract, 5 gof pepton and 2 g of glucose per liter) in a shaker incubator at 45° C.and 180 rpm for 29 hours and then transferred to a fermentor filled with3.8 liter of the same medium and grown at 45° C. under a 180 rpmstirring at a regulated pH of 6.8. After 91 hours of culture,sporulation was provoked by lowering pH to 4.4 while adding 40 ml of a10 g/l manganese sulfate solution. Dry weight in the solution wasestimated to 10.8 g/l as a result of 5 pairs of measurements. Asenumerated by the internally modified malachite green method, about 50%of the bacteria were sporulated after 72 hours at 45° C. The 4 litersthus produced were mixed with the 3 liters produced in the firstproduction test. It was centrifuged to obtain a 2.4% w/w dry weightaqueous spore solution.

MJ1 and MJ2 solutions were then mixed together to obtain a bacterialsolution MJ containing 2.4 weight percent of spore dry mater.

2) Extrusion Step

2 mixtures of polylactic acid PLA (recycled polylactic acid, Futuramat),wheat native flour (blé La Dorée®, AMO) and MJ solution were extrudedinto a co-rotating extruder Clextral BC21 (L=600 mm, L/d=24) with thefollowing conditions:

-   -   Extrusion temperature zones: 20/80/140/170 iso    -   Screw speed: 250 rmp    -   Torque: 40%    -   Internal pressure: 60-65 bar

The compositions of the 2 materials were the following:

-   -   L22B07: 77.5 w % PLA+22.5 w % wheat native flour    -   SPLA01: 90.2 w % L22B07+9.8 w % MJ solution        The compounds were dried in order to remove the residual water.

The two products obtained by granulation were injected into an Arburg100T press so as to form specific specimens. The injection conditionsare described in table 19 below :

TABLE 19 Conditions of injection of the granulated products Injection 1Injection 2 Reference L22B07V01 SPLA Temperatures ° C. 130-145-155- ″(Feed → Nozzle) 165-170 Imprint Multiple ″ specimen Assay speed (v401)m/min 25 ″ Assay volume (V403) cm³ 30 32.5 Counterpressure (p401) bar150 ″ Assay time (t402M) s 1.9 about 3.5 Injection pressure (p301) bar1750 ″ Injection flow rate (Q301) cm³/s 10 ″ Switching (V311) cm³ 5 ″Maintenance pressure (p321) bar 1650 ″ Maintenance flow rate (Q321)cm³/s 10 ″ Maintenance time (t321) s 20 ″ Cooling time (t400) s 20 ″Cycle time (t902M) s 48.6 40.9 Mattress (V321I) s 1.1 Variable: 0.1 to3.4 Ejector speed (v602) mm/s 50 Variable: 0.1 to 3.4 Ejector force(F601) kN 110 Variable: 0.1 to 3.4 Nozzle position Detached Detachedafter assay after assay Mold temperature ° C. 35 35   Injectability(marked from A to D) A A Color light dark Aspect A A Injected quantity40 Remarks Silpack = 1 Longer assay, irregular mattress3) Degradation Results

Degradation efficiencies were evaluated in Jolival water source. Foreach formulation, 4 specimens (initially weighted) were immersed in awater tank. The tanks were placed at 32° C. in a ventilated enclosure.

A sample was then taken after 1, 3, 6 and 9 months and weighed (afterdrying it at 103° C. for 48 h). Degradability was measured by mass loss.

TABLE 20 Weight loss of polymer/spore alloys Weight loss (w %) after xmonths of immersion L22B07 SPLA1 0 0 0 1 2.2 12.2 3 7.2 19.3 6 11.3 20.79 14.9 21.3

The introduction of spores into a PLA-based material is stronglyincreasing its degradability.

The invention claimed is:
 1. A process for preparing apolymer/biological entities alloy, comprising a step of mixing a polymerand biological entities, said biological entities degrade said polymer,wherein said alloy is prepared during a heat treatment performed at atemperature T above room temperature, at which the polymer is in apartially or totally molten state, said biological entities being ableto degrade said polymer in said alloy, and wherein said polymer ispolycaprolactone and said biological entity comprises a lipase.
 2. Theprocess as claimed in claim 1, wherein said temperature T is between theglass transition temperature and the melting point of said polymer. 3.The process as claimed in claim 1, wherein said temperature T is themelting point of said polymer.
 4. The process as claimed in claim 1,wherein said heat treatment consists of an operation chosen fromextrusion, injection-molding, thermoforming, rotary molding,compression, calendering, ironing, coating, stratification, expansion,pultrusion, extrusion blow-molding, extrusion-swelling andcompression-granulation.
 5. The process as claimed in claim 4, whereinsaid heat treatment consists of an extrusion operation.
 6. The processas claimed in claim 1, wherein the biological entities/polymer weightratio is between about 0.1% and about 10%.
 7. The process as claimed inclaim 1, wherein the biological entities/polymer weight ratio is about2%.
 8. The process as claimed in claim 1, wherein the lipase is selectedfrom the group consisting of lipase PS from Pseudomonas cepacia, lipaseAK from Pseudomonas fluorescens, lipase B from Candida antarctica, andmixtures thereof.
 9. The process as claimed in claim 1, wherein saidlipase is an amanolipases PS from Pseudomonas cepacia.
 10. Apolymer/biological entities alloy that may be obtained via the processas claimed in claim 1.